The experiments conducted under aim 1 have demonstrated that even relatively mild GVHD can diminish quantitative T cell immune responses to vaccination and functional immune responses to tumors expressing vaccine-targeted antigens. This work has been published (Capitini et al, Blood, 2009) and demonstrated the importance of preventing GVHD if BMT is to be optimized as a platform for immunotherapeutic approaches targeting malignancy. Under aim 2, we have established that inhibition of interferon gamma signaling can prevent the development of GVHD (Capitini et al, Blood 2009). Reduction in GVHD severity with disruption of interferon gamma signaling on T cells was not surprising given the known importance of this cytokine in GVHD but this approach results in immunodeficiency. The novel finding in these studies was that selective loss of interferon gamma receptor on bone marrow-derived non-T cells also prevented GVHD mediated by T cells with intact interferon gamma signaling and did so with preservation of qualitative and functional responses to vaccines. We have also established the extracorporeal photopheresis, a modality currently being used in the clinic to treat GVHD, also prevented GVHD with preserved vaccine response via modulation of IL-10 production in DC populations (Capitini et al, Biology of Blood and Marrow Transplantation, 2011). We next studied whether other components of the interferon gamma pathway could be targeted in donor bone marrow to prevent GVHD. Using bone marrow deficient in STAT1, a transcription factor necessary for interferon gamma signaling, we have confirmed that interference with this pathway in bone marrow-derived cells can prevent GVHD with preserved immune competence. To identify the relevant bone-marrow-derived cell population, we have selectively targeted STAT1 by generating mice with a floxed STAT1 gene (obtained from Dr. Lothar Hennighausen) that express the Cre recombinase under non-T cell promoters (CD11c (DC expression), lysozyme (on all phagocytic cells), and CD19 (B cell expression). In recipients of bone marrow from these donors, the STAT1 gene (and, thus, interferon gamma signaling) can be ablated in selective cell populations. Selective loss of STAT1 in all of these cell populations was not sufficient to prevent GVHD. Interestingly, further assessment of DC reconstitution in recipients of allogeneic STAT1 deficient bone marrow demonstrated expanded plasmacytoid DC (pDC) populations. Further more, we have confirmed that STAT1 remained intact in all of the Cre floxed STAT1 mouse strains generated thus far. We have obtained pDC-depleting antibodies (from Dr. Giorgio Trinchieri) to confirm that the expanded pDC populations are responsible for the GVHD protection observed in recipients of STAT1-deficinet bone marrow. Preliminary studies indicate that this is case. Based on these findings we have begun to analyze the characteristics of the expanded pDCs mediating GVHD resistance. Preliminary studies indicate that there is an increase in the frequency of CD9 negative pDCs, reported to be tolerogenic. In addition, expression of S100A8 and S100A9, genes associated with a suppressive phenotype in myeloid cells, is increased in pDCs from STAT1 deficient bone marrow recipients. Using S100A8/S100A9 deficient mice (obtained from Dr. Dimitri Gabrilovich through the Mackall laboratory) we have confirmed that loss of these genes in donor bone marrow increases the severity of GVHD. Aim 3 is in the initial stage. We have obtained selective Stat1 inhibitors and are beginning to test these in vitro. In addition, we used off-target inhibition of STAT1 by exenatide, a drug used in patients with diabetes, to demonstrate reduction of GVHD severity in our murine models.